The present invention relates to novel yeast strains which express cytochrome P450 activity, and to their use. It relates, in particular, to yeast strains which are able to produce a system of human cytochrome P450 enzymes, and to the plasmids which are used for constructing these strains.
The P450 cytochromes constitute a superfamily of membrane enzymes. These enzymes are monooxygenases which are involved, more specifically, in the metabolism of xenobiotics and drugs.
They are used, in particular, for:
diagnosing in vitro the formation of toxic or mutagenic metabolites by the human hepatic metabolism of natural or artificial xenobiotic molecules (pollutants, drugs or additives). This diagnosis is of prime importance for developing new pharmaceutical molecules,
identifying and destroying toxic or pollutant molecules from the environment, and
producing metabolites.
Because of their involvement; at one and the same time, in these detoxification processes and these toxicity phenomena, these proteins have been studied intensively (Guenguerich, 1988).
Nevertheless, these studies have rapidly come up against difficulties such as that of studying individual forms of P450 cytochromes. Heterologous expression systems have therefore been developed to overcome these problems.
The use of mammalian cells as hosts for heterologous expression has been developed since 1986 (Zuber et al., 1986). While these systems have the advantage of being closely related to hepatic cells (main location of the P450 cytochromes), they unfortunately suffer from low levels of expression.
While prokaryotic hosts, such as bacteria, admittedly enable substantial quantities of correctly folded cytochrome P450 to be obtained (Barnes et al., 1991), modifications of the 5xe2x80x2-terminal expressed part of the DNA, which cannot be circumvented, are observed with this type of host (Doehmer and Greim, 1992).
On the other hand, it is very particularly advantageous to choose eukaryotic hosts of the yeast type: this organism makes it possible to achieve conditions which are similar to those of human hepatic cells and gives rise to a high level of protein expression. Furthermore, yeast possesses, in endogenous form, all the enzymic machinery which is required for expressing membrane proteins of the cytochrome P450 type and their associated enzymes; thus, yeast has available a cytochrome b5 and an NADPH-cytochrome P450 reductase, i.e. two enzymes whose presence is required for cytochrome P450 to function.
Yeast therefore offers an advantageous solution to the different problems (Oeda K. et al., 1985; Pompon, 1988), since, with this organism:
the N-terminal sequences of the proteins which are expressed do not have to be modified (as is the case with expression in bacteria)
reasonable quantities of heterologous cytochrome P450 are obtained for various biochemical and structural studies,
a system of associated enzymes already exists in the organism.
Yeasts which have been specially studied for expressing heterologous proteins and which may in particular be mentioned are Kluyveromyces, Pichia, Hansenula, Candida and Saccharomyces, whose genome structures are well known. Various systems for expressing cytochrome P450 in yeasts have been described in the literature.
In strains termed first generation strains, P450 cytochromes have been expressed from plasmids and use the NADPH-cytochrome P450 reductase and the cytochrome b5 which are endogenous to yeast as electron donors (Pompon, 1988; Cullin and Pompon, 1988).
A first improvement of this system gave rise to strains termed second generation strains in which, yeast cytochrome P450 reductase was overexpressed (under the control of the GAL10-CYC1 promoter) and a human cytochrome b5 was coexpressed (patent WO93/02200 and Truan et al., 1993). These strains thus made it possible to obtain recombinant cytochrome P450 enzymic activities which were from 5 to 60 times greater in the isoform than in the starting strain.
Nevertheless, the existing systems are not entirely satisfactory: either they do not enable adequate expression of the proteins to be obtained, or the proteins which are obtained are not sufficiently similar to the human system.
The specific objective of the present invention is to propose a third strain generation which does not suffer from the abovementioned drawbacks. Unexpectedly, the Applicant demonstrated that it was possible simultaneously to replace both the yeast NADPH-cytochrome P450 reductase and the yeast cytochrome b5 with their human homologues. This is all the more surprising since simultaneous disruption of these two genes was known to be lethal in yeast and, until now, it has not been possible to obtain a viable strain in which these two genes are deleted.
In the strains which are claimed, the yeast cytochrome P450 reductase and/or the yeast cytochrome b5 have been replaced with their human homologue(s). This, very advantageously, makes it possible to create a system which is very similar to hepatic cells, given the fact that the whole multienzyme system is then of the same nature.
This novel system makes it possible to study the effect of the nature of the redox partners of the P450 cytochromes which are expressed as well as the stoichiometries which are required to obtain cytochrome P450 activities which are comparable to those which exist in liver.
The invention therefore relates, initially, to a genetically modified yeast strain which is characterized in that:
Firstly, the genes encoding the endogenous cytochrome b5 and the endogenous NADPH-cytochrome P450 reductase have been inactivated,
Secondly, it comprises a nucleic acid which encodes human NADPH-cytochrome P450 reductase,
Thirdly, it comprises a nucleic acid which encodes human cytochrome b5.
The nucleic acids which are used for integrating genes encoding human cytochrome b5 and human reductase into the strain are preferably cDNAs. The cDNAs containing the totality of the sequences encoding these two proteins have been isolated and sequenced (in the case of human reductase, see S. Yamano et al. Mol. Pharmacol. 1989 Vol. 36: 83-8, and, in the case of human cytochrome b5, see M. Miyata et al. Pharmacol. Res. 1989 Vol. 21: 513-20).
Very preferably, also, the selected yeast is Saccharomyces cerevisiae. 
Within the meaning of the present invention, an inactivated gene is understood as being a gene which has been rendered incapable of encoding its natural protein. The inability of the said genes to encode their natural proteins can be manifested either by the production of a protein which is inactive due to structural or conformational alterations, or by the absence of production, or by production of the natural protein at an attenuated level.
Various methods can be used to inactivate the native genes:
total or partial deletion of the gene. Deletion is understood as being any removal of the gene under consideration. This removal can be of a part of the region encoding the protein and/or of all or part of the transcription promoter region,
one or more point mutations in the gene. The mutations can be obtained by treatment with chemical mutagenic agents (such as alkylating, bialkylating or intercalating agents) or with physical mutagenic agents (X, gamma or ultraviolet rays), or by means of site-directed mutagenesis,
a mutational insertion due to the action of restriction enzymes, which interrupt the reading frame of the gene and inactivate the latter, and/or,
a gene disruption, for example in accordance with the protocol initially described by Rothstein [Meth. Enzymol. (1983)202]. In this case, the integrity of the coding sequence, will be disrupted in order to enable the wild-type sequence to be replaced, by means of homologous recombination, with a sequence which encodes the corresponding human protein.
According to the present invention, preference is given to using the method of gene disruption, as described below.
Various solutions are conceivable for transforming the claimed strains with a view to causing them to express the human enzymes according to the invention; on the one hand, it is possible to transform a wild-type strain, one of whose genes has been inactivated, with a replicative plasmid which contains the nucleic acid encoding the corresponding human protein. In this case, the nucleic acid is not integrated into the genome of the yeast.
On the other hand, it is possible to integrate a nucleic acid, in the form of a cDNA which encompasses the sequence encoding the human protein in question, into the genome of the yeast. In this case, the integration can be effected either into a known locus on this genome corresponding to a marker gene, thereby altering neither the reproductive properties of the yeast nor its viability, or at the site occupied by the inactivated native gene.
Both the nucleic acid encoding the reductase and the nucleic acid encoding the cytochrome b5 can be introduced into the strain using one of these methods.
According to the present invention, and in order to improve the stability of the strain and to achieve more favourable conditions, preference is given to choosing the embodiment which consists in integrating the nucleic acid encoding human NADPH-cytochrome P450 reductase and/or the nucleic acid encoding human-cytochrome b5 into the genome of the yeast, with a preferred embodiment of the present invention being to integrate these nucleic acids into the site of, and in place of, the endogenous genes.
According to another preferred embodiment of the present invention, the gene encoding human cytochrome b5 is integrated into an intergenic site for a marker gene, in particular into the SPL1/leu2 intergenic site.
The invention also relates to a strain which is characterized in that the nucleic acid encoding human cytochrome b5 is integrated into the genome.
A particular embodiment of the invention consists in introducing two cytochrome b5 copies into the transformed strain.
Another problem encountered by the Applicant is that of expressing human genes in yeast at an adequate level.
For this, it is advantageous for these genes to be placed under the control of a yeast promoter which enables them to be expressed. These yeast promoters can either be inducible or be constitutive. In the present application, a constitutive promoter is understood as being a promoter whose expression is constant under the standard culturing conditions. According to the present invention, at least one of the human genes is under the control of a constitutive yeast promoter.
This promoter is selected from the known promoters. The promoters of the genes for isocytochrome C1 (CYC1), alcohol dehydrogenase (ADH1), transcription elongation factor (TEF), yeast glyceraldehyde phosphodehydrogenase (GAPDH below) and yeast phosphoglycerate kinase (PGK below) can, for example, be used.
The promoter is preferably selected from the promoter of the gene for yeast glyceraldehyde phosphodehydrogenase, the promoter of the gene for yeast phosphoglycerate kinase and the endogenous promoter of yeast cytochrome b5. It should be pointed out that, in a particular embodiment of the invention, the gene encoding human cytochrome b5 is under the control of the endogenous Yb5 promoter.
The inducible promoter is preferably selected from the GAL10 and CYC1-GAL10 promoters.
It was previously not possible to obtain haploid strains which exhibited the characteristics which are of interest to us, that is inactivation of the abovementioned endogenous genes and replacement of these genes with nucleic acids encoding the corresponding human genes. There was no alternative but to use the diploid form. One of the advantages of the present invention is that of being able to work with haploid yeasts, thereby making it possible to achieve a greater degree of stability and to avoid unwanted recombinations.
According to a preferred embodiment of the invention, the strains are therefore characterized in that they are haploid.
The strains according to the invention possess at least one nucleic acid encoding human cytochrome P450. The said nucleic acid will preferably be integrated on a plasmid. The customary techniques can be used to transform the humanized yeast strains according to the invention with a plasmid for expressing any cytochrome P450, provided that the selection markers of the said plasmid are compatible with the yeast strains which have been developed. In particular, such plasmids can be obtained by using the techniques of the art to clone the coding sequence of a cDNA encoding any human cytochrome P450 into the cloning polylinker of the plasmid pYeDP60 (see materials and methods).
The cytochrome P450 can be selected, in particular, from the human P450 cytochromes 1A1, 1A2, 1B1, 2C8, 2C9, 2C18, 2C19, 2E1, 3A4 and 3A5. The humanized strains according to the present application exhibit indisputable advantages for expression as compared with the wild-type yeasts and as compared with the previously developed recombinant strains.
The invention also relates to a yeast strain according to the invention which is additionally caused to express the monooxygenase activity of a human cytochrome P450 which is carried by a plasmid.
Another embodiment of the invention consists in causing the previously described additional copy of the nucleic acid encoding cytochrome b5 to be incorporated into a plasmid and, in particular, to be incorporated into the plasmid which already contains a copy of the nucleic acid encoding the cytochrome P450. This is because it is particularly advantageous, for obtaining optimum activity of the P450, to be able to achieve a relative molar stoichiometry of at least 1/1 in the levels at which the human cytochrome b5 and the human P450 are expressed. The genomic integration of a single copy of the cytochrome b5 gene can, under certain circumstances, prove to be inadequate from the viewpoint of the high level of expression of the P450, as results from its expression from a multicopy plasmid according to the invention. Thus, the present-invention makes it possible to improve still further the efficacy of the system by describing the construction of a series of plasmids which carry, at one and the same time, a cassette for expressing the cytochrome P450 of interest and a cassette for expressing cytochrome b5. The specific structure of these plasmids makes it possible to achieve stable expression which is at a high level and where there is adequate stoichiometry between the two cytochromes. These plasmids are compatible with all the strains described in the application. Their use can also be extended to other strains.
The preferred object of the present invention is to develop a yeast strain which exhibits all the previously described characteristics. Several intermediates which were constructed for the purpose of achieving this strain are described.
In the present invention, the starting strains are preferably those which are described in the literature, in particular:
the strain W(xcex94B), which is described by Truan et al., and in which the gene encoding the yeast cytochrome b5 (termed Yb5 below) has been disrupted. In order to achieve this, a vector is constructed which possesses the HIS3 marker gene integrated into a restriction site in the cytochrome b5 gene. This vector is used to transform a diploid HIS3xe2x88x92 strain. The recombinants are selected,
the strain W(R), in which the gene encoding yeast reductase has not been inactivated,
and the strain W(hR), which is obtained from a W(Rxcex94) strain by means of transformation with the vector pUP81 (FIG. 1). In this strain, the gene encoding the inactivated yeast reductase (termed YRED below) has been replaced by inserting a cassette which contains the inducible promoter and the sequence encoding human reductase (termed HRED below).
These strains are crossed and then sporulated, and the haploid W(hR,xcex94B) strains are selected which are deleted for Yb5 and YRED and which express human reductase under the control of the GAL10-CYC1 promoter.
In this respect, the invention also relates to a strain which comprises a nucleic acid encoding human NADPH-cytochrome P450 reductase under the control of the GAL10-CYC1 promoter and whose genes encoding yeast cytochrome b5 and yeast NADPH-cytochrome P450 reductase have been inactivated (strain W(hR,xcex94B)).
Another strain which is preferred according to the invention consists of a strain which is identical to the preceding strain but in which the inducible GAL10-CYC1 promoter has been replaced with the GAPDH promoter.
This replacement can be effected by transforming with the plasmid pAB2 (FIG. 4), which is constructed from pUP81 (FIG. 9) and which contains a cDNA encoding HRED under the control of the constitutive GAPDH promoter. The transformants are selected using the method described in patent WO94/01564. The strain which is obtained is designated W(GhR,xcex94B). In this strain, the sequence encoding human reductase is under the control of the constitutive yeast GAPDH promoter, and the yeast cytochrome b5 and YRED are inactivated.
The invention also relates to a strain which is characterized in that the nucleic acid encoding human NADPH-cytochrome P450 reductase is under the control of the promoter of the gene for yeast glyceraldehydephosphodehydrogenase.
Another strain which is preferred according to the invention is obtained from the W(hR,xcex94B) strain by means of transformation with the vector pAB3 (FIG. 6 and 12), which contains the sequence encoding human cytochrome b5.
Yeasts are selected which have integrated this vector. The resulting strain is termed W(hR,hb5). This strain possesses the two sequences which encode human proteins.
In order to construct a strain which is particularly advantageous according to the invention, the strain W(GhR,xcex94B) is transformed with the plasmid pAB3 in the same manner as before. This results in a strain, W(GhR,hb5), which possesses the properties of the two preceding strains, namely that it expresses the gene encoding human reductase under the control of the yeast GAPDH promoter and that it can also express the gene encoding human cytochrome b5 under the control of the yeast pYb5 promoter.
In a manner which is equally well preferred, the Applicant has constructed a strain from the W(hR,xcex94B) strain. This strain is transformed with the plasmid vector pAP1 (FIG. 8 and 13), which has been previously linearized with a restriction enzyme. The transformants are selected which have integrated the sequence encoding human cytochrome b5 under the control of the yeast PGK (phosphoglycerate kinase) promoter, carried by pAP1 into the leu2/SPL1 intergenic site of the yeast chromosome (FIG. 14).
In the Applicant""s nomenclature, this strain has the designation W(hR,Lhb5). It is able to express the sequence encoding human reductase under the control of the GAL10-CYC1 promoter and the sequence encoding cytochrome b5 under the control of the yeast PGK promoter.
The invention also relates to a yeast strain which is characterized in that it comprises at least one nucleic acid which encodes human cytochrome b5 under the control of the promoter of the gene for yeast phosphoglycerate kinase.
Very particular preference is given to the following strain. The starting strain is the strain W(GhR,xcex94B), which is transformed with the linearized pAP1 vector. The clones are selected which have integrated the sequence encoding human cytochrome b5 under the control of the yeast PGK promoter into the abovementioned leu2/SPL1 chromosomal site.
This thereby results in a strain, W(GhR,Lhb5), which comprises the sequences encoding the two human genes under the control of two constitutive yeast promoters.
Another part of the subject-matter of the invention is characterized in that a strain comprises, at one and the same time:
the nucleic acid encoding human NADPH-cytochrome P450-reductase under the control of the promoter of the gene for yeast glyceraldehyde phosphodehydrogenase,
and the nucleic acid encoding human cytochrome b5 under the control of the promoter of the gene for yeast phosphoglycerate kinase.
Another embodiment of the invention consists in starting with the strain W(R) and transforming it with the pAP1 vector; after selection, the strain W(R,Lhb5,Yb5) is obtained.
Preference is given to starting with the strain W(hR), which is then transformed in the same manner; after selection, the strain W(hR,Lhb5,Yb5) is obtained.
The strains which have been constructed in accordance with the present invention make it possible to develop processes which are directed towards evaluating the toxicity of the metabolites which arise from the degradation of novel chemical molecules by the cytochrome P450 enzyme system.
The invention also relates to a process for evaluating the toxicity of a compound, characterized in that:
the said compound is brought into contact with a yeast according to the invention or with an enzyme preparation which is derived from such a yeast, and the toxicity of the metabolites which are produced is analysed.
The present invention furthermore makes it possible to obtain an enzyme complex which is very similar to that which exists in human hepatic cells. This provides the possibility of working in vitro under favourable conditions for expressing human enzymes. Thus, it is possible to determine the metabolites which will result, in man, from the degradation of novel chemical compounds by the cytochrome P450 complex.
The invention also relates to a method for determining in vitro the human metabolites of a chemical compound, characterized in that:
the said compound is brought into contact with a yeast according to the invention, or with an enzyme preparation which is derived from such a yeast, and the metabolites which are produced are identified.
The present invention is described in more detail with the aid of the examples which follow and which should be regarded as being illustrative and not limiting.